Methods and apparatus for kidney dialysis and extracorporeal detoxification

ABSTRACT

The present disclosure relates to a dialysis apparatus comprising a membrane having at least one protein from the lipocalin family bound thereon. The disclosure further relates to methods of removing non-polar, hydrophobic and/or protein bound uremic toxins from a target subject utilizing the dialysis apparatus described herein as well as methods of extracorporeal detoxification.

STATEMENT OF PRIORITY

This application claims the benefit, under 35 U.S.C. § 119(e), of U.S.Provisional Application Ser. Nos. 61/919,024, filed Dec. 20, 2013 and62/031,257, filed Jul. 31, 2014, and is a continuation-in-part ofInternational Application No. PCT/US2014/071212, filed Dec. 18, 2014,the entire contents of each of which are incorporated by referenceherein.

TECHNICAL FIELD

The present disclosure relates to a method and dialysis apparatusdesigned to facilitate the clearance of hydrophobic, protein bounduremic toxins from subjects with impaired kidney function and/oraffected by drug overdose.

BACKGROUND

Hemodialysis, hereinafter referred to as kidney dialysis, or simply“dialysis,” is a medical procedure that is performed on target subjects,for example humans, (and also, on a smaller scale, pet animals), toremove accumulated waste and toxins from the blood in a similar mannerto a functioning kidney. When a person or animal's kidneys cease tofunction properly due to one or more of a number of acute or chronicdiseases or conditions (e.g., diabetes, glomerulonephritis andhypertension are commonly recognized medical conditions that areassociated with the development of renal failure), toxins accumulate inthe bloodstream.

Failure to remove such accumulated waste and toxic compounds—primarilyurea, uric acid and its analogues, and other nitrogenous compounds suchas creatinine; and excess amounts of elements such as potassium,phosphorous, sodium, chloride and other minerals from the blood resultsin deterioration of body tissues and organ systems, which eventuallyresults in death if untreated. Moreover, failure to remove such wasteand toxins may result in uremia, which is a clinical syndrome that inmany aspects resembles systemic poisoning. Almost every organ system inthe body is affected by uremic toxicity and the known uremic clinicalsymptoms and side effects include, but are not limited to, fatigue,anemia, itching, peripheral neuropathy, gastrointestinal disordersincluding nausea, vomiting, diarrhea, cardiovascular complicationsincluding accelerated coronary and peripheral vascular disease, leftventricular hypertrophy, cardiac fibrosis and accelerated rates ofarrhythmias. Conventional hemodialysis is capable of treating uremicsymptoms that arise from “water soluble” non-hydrophobic uremic toxins,but dialysis is very ineffective in treating symptoms that arise fromhydrophobic, protein-bound uremic toxins.

Generally, dialysis interposes a semi-permeable membrane between aflowing stream of blood and an appropriate rinsing solution. Byconvective and diffusive transport, the composition of body fluidsapproaches that of the dialysis solution. Dialysis may be performed in ahospital setting or clinic; or in some cases, the target subject istrained to perform the procedure at home on an outpatient basis. Twoprimary types of dialysis are regularly performed—conventionalhemodialysis and peritoneal dialysis. In conventional hemodialysis, thetarget subject is connected (via an arteriovenous fistula, graft or bycatheter) to a dialysis machine. The dialysis machine functions to pumpthe contaminated blood from the target subject through a dialyzer, wherethe blood is filtered through a dialyzing solution, thereby lowering theconcentration of accumulated waste (e.g., urea), and thence returned tothe target subject. Current dialysis membranes and technology arecapable of clearing water soluble uremic toxins, but exhibit limitedclearance of non-polar, hydrophobic, or protein bound toxins.

For example, p-cresol is an organic compound with the formulaCH₃C₆H₄(OH). It is a colourless solid that is a derivative of phenol andan isomer of o-cresol and m-cresol. A limitation of current dialysistreatment is the inability to remove all toxins from the bloodstreamduring a dialysis procedure. In particular, small hydrophobic compoundssuch as p-cresol, are known to build up and have the potential to causesevere toxicity. Current dialysis membranes and technologies are able toclear water soluble uremic toxins, but have limited clearance ofnon-polar protein bound toxins. Non-polar uremic toxins include, but arenot limited to, p-cresol, p-cresol sulfate, and indoxyl sulfate. Thus,many target subjects, including patients undergoing kidney dialysisprocedures, especially target subjects with advanced kidney disease havean accumulation of p-cresol in the plasma.

Further, p-cresol is derived from phenylalanine metabolism and has oneof the highest plasma level of any known non-polar uremic toxins.Moreover, p-cresol, indoxyl sulfate and other protein bound uremictoxins have been linked to the development of vasculopathy, leftventricular hypertrophy, cardiac fibrosis as well as atrial andventricular arrhythmias.

Accordingly, what is needed is a method and/or an apparatus that may beused during a dialysis procedure to remove hydrophobic, non-polar uremictoxins, such as p-cresol, from the blood.

BRIEF SUMMARY

In particular embodiments, the disclosure relates to a dialysisapparatus that includes materials including, but not limited to,polysulfone and other materials that are used for filtering accumulatedtoxins of patients with impaired kidney function. The dialysis materialsin the present apparatus may be bound to one or more proteins in variouscombinations from the lipocalin family of proteins.

In other embodiments, the disclosure relates to a modified dialysiscartridge having at least one member of the lipocalin family. Theapparatus may include a member of the olfactory binding proteins (OBP)and any other member of the lipocalin family of proteins that arecovalently bound to dialysis filtering materials. The therapeuticpurpose of bound lipocalin proteins will be to facilitate the removalnon-polar, hydrophobic, water soluble, water insoluble and/or proteinbound blood toxins that accumulate in the plasma of patients withadvanced renal disease.

In further embodiments, the disclosure provides methods of removingnon-polar, hydrophobic and/or protein bound uremic toxins from a targetsubject comprising using a dialysis apparatus having materialsincluding, but not limited to, polysulfone and other materials that areused for filtering accumulated toxins of patients with impaired kidneyfunction. The dialysis materials may be bound to one or more proteins invarious combinations from the lipocalin family of proteins. Methods ofremoving non-polar, hydrophobic and/or protein bound uremic toxins froma target subject comprising using a dialysis apparatus may furthercomprise using a priming solution that is passed through the thecartridge prior to use. The solution includes one or more lipocalinsthat would attach to the dialysis cartridge membrane.

In some embodiments, the disclosure provides methods of preventing ortreating uremia including uremic clinical symptoms and side effects.

In some embodiments, the disclosure provides methods and apparatus forfacilitating clearance or removal of prescription and non-prescriptiondrugs from a subject.

In some embodiments, the disclosure provides methods and apparatus forfacilitating clearance or removal of prescription and non-prescriptiondrugs from a subject resulting from an overdose.

It is to be understood that both the foregoing general description andthe following detailed description present embodiments of the disclosureand are intended to provide an overview or framework for understandingthe nature and character of the disclosure as it is claimed. Thedescription serves to explain the principles and operations of theclaimed subject matter. Other and further features and advantages of thepresent disclosure will be readily apparent to those skilled in the artupon a reading of the following disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of a polysulfone membrane having at leastone lipocalin protein bound thereon.

FIG. 2 is another schematic drawing of a polysulfone membrane having atleast one lipocalin protein bound thereon.

FIG. 3 is a schematic drawing of a lumenal view of lipocalin proteinsbound to a polysulfone membrane.

FIG. 4 illustrates schematically a system for performing dialysis usingthe dialysis apparatus described herein.

DETAILED DESCRIPTION

Reference now will be made in detail to the embodiments of the presentdisclosure, one or more examples of which are set forth hereinbelow.Each example is provided by way of explanation of the apparatus and/ormethods of the present disclosure and is not a limitation. In fact, itwill be apparent to those skilled in the art that various modificationsand variations can be made to the teachings of the present disclosurewithout departing from the scope of the disclosure. For instance,features illustrated or described as part of one embodiment, can be usedwith another embodiment to yield a still further embodiment.

Thus, it is intended that the present disclosure covers suchmodifications and variations as found within the scope of the appendedclaims and their equivalents. Other objects, features and aspects of thepresent disclosure are disclosed in or are apparent from the followingdetailed description. It is to be understood by one of ordinary skill inthe art that the present discussion is a description of exemplaryembodiments only and is not intended as limiting the broader aspects ofthe present disclosure.

The present disclosure relates generally to a dialysis apparatus thatincludes at least one protein from the lipocalin family. In someembodiments, the dialysis apparatus is a dialysis cartridge that hasbeen modified to have at least one protein from the lipocalin familycovalently bound to a membrane located within the blood side of thecartridge. In some embodiments, the at least one protein from thelipocalin family is bound to a polysulfone membrane. Additionally, thedisclosure relates to methods of removing non-polar toxins, hydrophobictoxins and/or protein bound toxins from the blood of a target subject byutilizing a dialysis apparatus that includes at least one protein fromthe lipocalin family.

In general, a dialysis cartridge is a device comprising plastic andother materials that is designed to rapidly process large volumes ofblood (e.g. 500 mls/min) from patients with no kidney function through achamber that is composed of modified polymers (polysulfone orpolyacrylamide materials) that can function to filter the blood ofexcess electrolytes waters and toxins generated by cellular metabolism(e.g., uremic toxins). These polymers are generally manufactured in theform of hollow fibers or arranged in stacked sheets (e.g., platedialyzers) in a manner that allow blood to be exposed under varyingdegrees of pressure to one side of the polymer material. Pores oropenings within the polymer allow for excess electrolytes, water anduremic toxins to flow through “pores” within the polymers and into acomplex electrolyte solution (e.g., dialysate) flowing on the other sideof the polymer. This structure allowing movement of electrolytes, waterand uremic toxins with the blood of across a semi-permeable polymermembrane and into a dialysate solution is generally understood in theart to encompass the definition of a dialysis cartridge. A dialysisapparatus can include the use of large gauge hollow needles that areused to access either a dialysis fistula or catheter where blood fromthese needles is pumped through a dialysis cartridge at high pressure.This dialysis apparatus (as shown in the figures) can pump in adirection opposite to the flow of blood through the cartridge. Thisblood dialysate interface can maximize the process of movement of uremictoxins through the filter into the dialysate and out of the body. Thus,the dialysis apparatus may accomplish the process of clearance of excesselectrolytes, water and uremic toxins.

The target subject is connected to a hemodialysis machine and the targetsubject's blood is pumped through the machine. Blood is accessed inpatients requiring treatment with the present inventive concept by acentral dialysis catheter, dialysis arteriovenous fistula or dialysisarterial graft. Dialysis procedures separate elements in a solution byutilizing diffusion across a semi-permeable membrane (diffusive solutetransport) down a concentration gradient. Waste, toxins, and excesswater are removed from the target subject's blood and the blood isinfused back into the target subject. Each dialysis treatment typicallylast between 3-4 hours and three times per week.

The hemodialysis technologies can be designed and configured for on-siteor at-home dialysis treatments. Furthermore, dialysis apparatus can beused in portable dialysis treatment devices such as, for example,wearable artificial kidneys in which a target subject may move freelyduring dialysis.

The present disclosure provides a dialysis apparatus for providingclearance of non-polar uremic toxins in a target subject that includespatient's home hemodialysis, nocturnal hemodialysis and the potential of“wearable” portable dialysis machines.

In some embodiments, the present disclosure is directed to a dialysisapparatus comprising a body having an inlet and an outlet and definingan interior, the interior including a layer comprising at least onepolysulfone membrane having at least one protein from the lipocalinfamily bound thereto. In some embodiments, the dialysis apparatus isconstructed and arranged so that a fluid, such as blood or plasma,entering the apparatus contacts the at least one lipocalin protein uponentering the apparatus and before exiting the device through the outlet.In some embodiments, the dialysis apparatus comprises a polysulfonemembrane having a plurality of proteins from the lipocalin family boundthereto. Still in some embodiments, where the polysulfone membraneincludes a plurality of proteins, the plurality of proteins may be thesame protein selected from the lipocalin family, or the plurality ofproteins may comprise a combination of different types of proteinsselected from the lipocalin family. In particular embodiments, themembrane is covalently bound to the at least one protein from thelipocalin family. Further, the membrane is specifically bound to the atleast one protein from the lipocalin family.

Generally, the family of lipocalin proteins refers to a family ofproteins that transport small hydrophobic molecules. Non-limitingexamples of lipocalin family proteins include, but are not limited tothe following, fatty acid binding proteins, olfactory binding proteins,and retinol binding proteins. Each protein within the lipocalin familyshares limited regions of sequence homology and a common tertiarystructure architecture. Generally, the tertiary structure comprises aneight stranded antiparallel beta-barrel and encloses an internalhydrophobic ligand binding domain. Additionally, the lipocalin family ofproteins includes a 3₁₀-like helix that is located on the inferiorsurface of the beta-barrel. The short 3₁₀-helix, helps to close off oneend of the barrel of the lipocalin protein thus allowing capture of avariety of hydrophobic molecules. The beta-barrel and 3₁₀ helix arecommon to proteins within the larger lipocalin family.

Additionally, the structure of the lipocalin fold is dominated by eightbeta-strands, which together form a calys- or cup-shaped antiparallelbeta-barrel, which encloses an internal ligand binding site. (See,Flower et al., “Structure and sequence relationships in the Lipocalinsand related proteins.” Protein Science (1993), 2, 753-761)).

More specifically, the structure of the L1 includes a large omega-loopand generally forms a lid folded back to cap or close the internalligand-binding site. The confirmation of this loop varies in detailbetween different proteins of the lipocalin family but maintains itsoverall shape, size, and position. The L1 loop serves to close theinternal ligand-binding site, which serves to enclose the ligand in theprotein and prevent it from becoming detached from the protein. The 3₁₀alpha-helix then closes off the other end of the barrel. Accordingly, inconjunction these two particular components, the L1 loop and the 3₁₀alpha helix of the lipocalin protein encase the ligand.

Accordingly, in some embodiments herein, proteins having a similarbeta-barrel structure, 3₁₀ helix and/or L1 loop, may be utilized in thepractice of the present disclosure. Moreover, proteins including abeta-barrel structure that are capable of binding hydrophobic ligandsmay be utilized in some embodiments in the practice of the presentdisclosure. As will be understood by one of ordinary skill in the art,proteins that include any molecular or structural modification of thebeta-barrel, 3₁₀ helix structure of the lipocalin family of proteins orthe L1 loop at the opening of the ligand binding domain of the lipocalinfamily of proteins, may be utilized in the practice of the presentdisclosure. Further, in some embodiments, at least one lipocalin proteinmay include a genetically modified lipocalin protein. Potential geneticmodifications include modification of the L1 loop or the 3₁₀ alphahelixes at either end of the protein, as well as the internal structureof the protein itself Moreover, in some embodiments, the at least onelipocalin protein may include a genetically modified lipocalin proteinthat has been modified so as to exhibit increased binding affinity ofuremic toxins, and/or increase amount of protein binding (i.e. improvedstoichiometry), both of which would improve the clearance of uremictoxins when utilized in practice of the present disclosure. In someembodiments, the at least one lipocalin protein may be a geneticallymodified lipocalin protein that is genetically modified by any processcurrently known in the art or later developed.

In some embodiments, the at least one lipocalin protein used in thedialysis apparatus of the present disclosure may be comprised of, but isnot limited to, at least one or any combination of the followingproteins: alpha-1-microglobulin (protein HC), major urinary proteins,alpha-1-acid glycoprotein (orosomucoid), aphrodisin, apolipoprotein D,beta-lactoglobulin, complement component C8 gamma chain, crustacyanin,epididymal-retinoic acid binding protein (E-RABP), insectacyanin,odorant binding protein (OBP), human pregnancy-associated endometrialalpha-2 globulin (PAEP), probasin (PB), a prostatic protein,prostaglandin D synthase, purpurin, Von Ebner's gland protein (VEGP),and lizard epididymal secretory protein IV (LESP IV). In particularembodiments, the at least one or any combination of the followingproteins includes: FABP1, FABP2, FABP3, FABP4, FABP5, FABP6, FABP7,LCN1, LCN2, LCN8, LCN09, LCN10, LCN12, OBP2A, OBP2B, RBP1, RBP2, RBP4,RBP5, RBP7, PAEPPERF15, PMP2, PTGDSAMBP, APOD, C8G, CRABP1, CRABP2UNX2541 ORM1 and ORM2. The source at least one or any combination ofproteins is not particularly limited. In some embodiments, the source ofthe proteins is mammalian, for example, such as canines, felines,bovines, caprines, equines, ovines, porcines, rodents (e.g. rats andmice), lagomorphs, primates (including non-human primates), etc. In oneembodiment, the proteins are human proteins.

In other particular embodiments, the at least one or any combination ofproteins includes: human lipocalin 1 (LCN1, for example, as set forth inGenBank/NCBI Accession Nos. NP_001239546.1, NP_002288.1, NP_001239547.1and/or NP_001239548.1); human lipocalin 2 (LCN2, for example, as setforth in GenBank/NCBI Accession Nos. AAH33089.1 and/or NP_005555.2);human lipocalin 8 (LCN8, for example, as set forth in GenBank/NCBIAccession Nos. AAQ81973.1, NP_848564.2, XP_005266115.1, XP_006717016.1,AAI32715.1 and/or AAI30466.1); human lipocalin 9 (LCN9, for example, asset forth in GenBank/NCBI Accession Nos. AAQ81975.1, NP_001001676.1and/or XP_006717169.1); human lipocalin 10 (LCN10, for example, as setforth in GenBank/NCBI Accession Nos. AAQ81976.1, AAI33046.1, and/orNP_001001712.2); human lipocalin 12 (LCN12, for example, as set forth inGenBank/NCBI Accession Nos. AAQ81977.1, NP_848631.2, XP_005266125.1,XP_005266126.1, XP_005266127.1, XP_005266128.1 and/or XP_005266129.1);human olfactory (odorant) binding protein isoform 2a (OBP2A, forexample, as set forth in GenBank/NCBI Accession Nos. AAH69563.1,NP_055397.1, NP_001280118.1, NP_001280122.1 and/or XP_006717150.1);human olfactory (odorant) binding protein isoform 2b (OBP2B, forexample, as set forth in GenBank/NCBI Accession Nos. AAQ89340.1,AAH98340.1, NP_055396.1, NP_001275916.1 and/or XP_006717149.1); humanretinol binding protein 1 (RBP1, for example, as set forth inGenBank/NCBI Accession Nos. NP_002890.2, NP_001124464.1 and/orNP_001124465.1); human retinol binding protein 2 (RBP2, for example, asset forth in GenBank/NCBI Accession Nos. NP_004155.2, XP_005247750.1and/or XP_006713785.1); human retinol binding protein 4 (RBP4, forexample, as set forth in GenBank/NCBI Accession Nos. NP_006735.2 and/orXP_005270080.1); human retinol binding protein 5 (RBP5, for example, asset forth in GenBank/NCBI Accession No. NP_113679.1); and/or humanretinol binding protein 7 (RBP7, for example, as set forth inGenBank/NCBI Accession No. NP_443192.1).

OBPs are members of the lipocalin family of proteins and are found inthe antennae of various insects. For the purposes of this disclosure,the terms “odorant binding protein” and “olfactory binding protein” maybe used interchangeably relating to OBP. Human OBP2A (NCBI Accession No.NP_055397.1), for example, is comprised of 170 amino acids, however thefirst 15 amino acids code for a signal peptide that is cleaved offduring protein synthesis. As such, the mature OBP2A peptide contains 155amino acids. Generally, OBP2A is an eight-stranded, antiparallel,symmetrical β-barrel fold, which has been rolled into a cylindricalshape. Inside the barrel of the OBP2A protein is a ligand binding site.While specific information regarding post-translational modification ofOBP2A is limited, there is a disulfide bridge formed between cysteineslocated at position 74 and 166 respectively. Further, serine 51 of ratOBP can be phosphorylated, but it is unknown whether this occurs inhumans. See Jonathan Pevsner S, Vivian Hou, Adele M. Snowman, andSolomon H. Snyder. Odorant-binding Protein: Characterization and LigandBinding J. Biol. Chem. Vol. 265, No. 11, Issue of April 15, pp.6118-6125, 1990.

Accordingly, in some embodiments, the dialysis apparatus comprises atleast one polysulfone membrane having at least one olfactory bindingprotein attached thereon. In some embodiments, the at least oneolfactory binding protein may be selected from, but is not limited tothe following: OBP2A, OBP2B and combinations thereof. In still someembodiments, the dialysis apparatus comprises at least one polysulfonemembrane having a plurality of olfactory binding proteins bound thereon.In some embodiments, the dialysis apparatus comprises a disposabledialysis cartridge.

In some embodiments, the at least one lipocalin protein may besynthesized and purchased from a commercial company that specializes inthe synthesis of complete proteins, for example, Bio-synthesis(Lewisville, Tex.). Further, in some embodiments, C-DNA vectors for oneor more of the lipocalin protein(s) are expressed in yeast culturesystem. To minimize degredation of the expressed lipocalin proteins,generally all yeast cultures include a cocktail of various proteaseinhibitors. In some embodiments, confirmation that a specific lipocalinprotein(s) has been expressed may be validated using SDS-PAGEelectrophoresis and immunoblotting of western blots. In someembodiments, the lipocalin protein(s) are at least 70%, 75%, 80%, 85%,90%, 95%, 99% or 100% free of contaminants. In some embodiments, thelipocalin protein(s) utilized in practice of the present disclosure willbe at least 90% pure.

In some embodiments, at least one protein from the lipocalin family maybe bound to the membrane by any method known in the art. For example, insome embodiments at least one lipocalin protein is bound to the membraneby a histidine tag. Any method known in the art for expressing alipocalin protein with a histidine tag and/or multiple histidineresidues may be utilized herein to attach the lipocalin protein to themembrane. In some embodiments, at least one lipocalin protein iscovalently bonded to a polysulfone membrane by any method known in theart. In some embodiments, as a result of the nature of the synthesis ofthe lipocalin protein(s) by the commercial company, the lipocalinprotein(s) will contain a histidine tag regardless of the cell type itis expressed in, that will be utilized in the binding of the protein tothe cartridge membrane. In any instance, the binding is specific bindingand not non-specific binding.

For example, in some embodiments, in order to bind the at least oneprotein from the lipocalin family, the vector encoding for a specificlipocalin protein will include a histidine tag. For example, in someembodiments, the carbolxylation reaction described, for example, inMockel et al., Journal of Membrane Science; 158: 63-75, 1999; and Guiveret al., British Polymer Journal; 23: 29-39, 1990 are hereby incorporatedby reference. Moreover, in some embodiments, the at least one lipocalinprotein is bonded to the polysulfone membrane by exposing the at leastone lipocalin protein including a histidine tag to a chelatedpolysulfone membrane in the presence of Li⁺ or some other chelatingmetal such as Ni²⁺ or Co²⁺ in the presence of dry ice. This process willallow for the binding of histidine, which is located on the at least onelipocalin protein, to the chelated polysulfone membrane.

In some embodiments, the at least one protein from the lipocalin familymay be bound (e.g., specific binding) to any suitable membrane known inthe art. In some embodiments, the membrane selected may include but isnot limited to any one or combinations of the following: polysulfone,polyethersulfone, AN69 (acrylonitrile membrane), and cellulose acetate.For example, in some embodiments the membranes used in the dialysisapparatus may include cellulose-based membranes, synthetic membranes andcombinations therefor. Non-limiting examples of cellulose-basedmembranes include: cellulose acetate, triacetate, and combinationsthereof Non-limiting examples of synthetic membranes include: polyamide,polysulfone, polyethersulfone, polyacrylonitrile, polymethylmethacylate,and combinations thereof.

In some embodiments, the at least one membrane may be polyethersulfone.In some embodiments, the polyethersulfone membrane may be chemicallymodified to chelate a covalent ion to the membrane surface as described.See also Kroll et al., Journal of Membrane Science; 299: 181-189, 2007.In addition to polysulfone, in some embodiments the membrane selectedmay be a membrane having a similar structure to polysulfone.

Briefly, FIG. 1 illustrates the interior of the body of the dialysisapparatus that includes at least one protein from the lipocalin family.As shown in FIG. 1, the polysulfone membrane 10 includes at least oneprotein from the lipocalin family 20 that is covalently bonded to thepolysulfone membrane 10 via a histidine tag 30. Further, the polysulfonemembrane, in some embodiments, includes a plurality of proteins from thelipocalin family 20. A further depiction of the interior of the body ofa dialysis apparatus of the disclosure is shown in FIG. 2. FIG. 3provides an illustration of the lumen of a polysulfone membrane havinglipocalin proteins bound thereto.

As shown in FIG. 4, a fluid waste stream 100 from a target subjectenters a fluid flow path 102 and enters the dialysis apparatus 104. Thedialysis apparatus 104 includes an inlet 105 and an outlet 106. Aspreviously described in reference to FIG. 1, the interior of thedialysis apparatus includes at least one protein from the lipocalinfamily (not shown in FIG. 4). In some embodiments, the dialysisapparatus 104 is a dialysis cartridge. The fluid waste stream 100 may ormay not have been treated prior to entering the dialysis apparatus 104.As the fluid waste stream passes through the dialysis apparatus 104, thenon-polar, hydrophobic, or protein bound uremic toxins are removed fromthe fluid waste stream 100 as they come into contact with the protein(s)from the lipocalin family that is bound to the interior of the dialysisapparatus 104. As a result of passing through the dialysis apparatus104, a clean fluid stream 114, void of or having a lower concentrationof non-polar, hydrophobic, or protein bound uremic toxins emerges fromthe outlet 106 of the dialysis apparatus 104. The clean fluid stream 114may have up to 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% lowerconcentration of non-polar, hydrophobic, or protein bound uremic toxins.The final clean fluid stream 114 then exits the dialysis apparatus 104via the outlet 106 and enters a fluid flow path 116 and then goes backto the target subject.

In some embodiments, the dialysis apparatus 104 may comprise additionalfilters, for example, in some embodiments, the dialysis apparatus 104may include a filtration membrane that is capable of retaining chargedspecies such as Ca2+, Mg2+, Na+ and proteins.

In certain embodiments, to improve the treatment capacity of thedialysis apparatus 104, the flow rate of the fluid waste stream 100should be sufficiently long enough to allow the non-polar, hydrophobic,or protein bound uremic toxins to bind with the proteins from thelipocalin family located within the dialysis apparatus 104. For example,in some embodiments the flow rate of the fluid waste stream 100 may befrom about 200 mls/min to about 600 mls/min. In still some embodiments,the flow rate of the fluid waste stream 100 may be from about 300mls/min to about 500 mls/min. Still in other embodiments, the fluidwaste stream 100 may be from about 350 mls/min to about 450 mls/min.

In some embodiments, the flow rate of the clean fluid stream 114 afterleaving the dialysis apparatus 104 and returning to the target subjectis from about 50 mls/min to about 300 mls/min. In still some otherembodiments, the flow rate of the clean fluid stream 114 is from about100 mls/min to about 200 mls/min.

Additionally, in some embodiments, ions and/or fluids can be replaced inthe stream, for example, through the addition of concentrated dialysiscomponents such as osmotic agents (e.g., dextrose, icodextrin, glucosepolymers, glucose polymer derivatives, amino acids), buffers (e.g.,lactate, bicarbonate) and electrolytes (e.g., sodium, potassium,calcium, magnesium) from a suitable fluid source.

In some embodiments, a pump may be used to propel the fluid waste streaminto the fluid flow path 102, through the dialysis apparatus 104, into asecond fluid flow path 116 and back to the target subject. Any pumpknown or used in the art may be utilized herein. In some embodiments afirst pump may be used to pump the fluid waste stream 104 from thetarget subject into the dialysis apparatus 104, while a second pump maybe utilized to pump the clean fluid stream from the dialysis apparatus104 back to the target subject. In still other embodiments, at least oneflow restrictor and/or at least one pump may be used to create pressuregradients. In this regard, the at least one flow restrictor and/or atleast one pump can provide a sufficiently high pressure to force fluidthrough the dialysis apparatus 104 and fluid flow paths 102 and 114.

In some embodiments, the clean fluid stream 114 may be passed through aclear cartridge 120 before re-entering the target subject. As shown inFIG. 4, in some embodiments, the clean fluid stream 114 may bypass theclear cartridge 120 and re-enter the target subject, and in otherembodiments, the clean fluid stream 114 enters a separate fluid flowpath 112 which allows the clean fluid stream 114 to pass through theclear cartridge 120 before re-entering the target subject.

Additionally, disclosed herein are methods of removing non-polar,hydrophobic, and/or protein bound toxins from a target subjectcomprising utilizing a dialysis apparatus wherein the dialysis apparatuscomprises at least one membrane having at least one protein selectedfrom the lipocalin family bound thereon. In some embodiments, the atleast one membrane is a polysulfone membrane. In some embodiments, themethods disclosed herein may comprise the steps of inserting a catheteror other suitable device into the target subject to remove blood fromthe target subject, passing or transferring the blood through a fluidflow path and into the dialysis apparatus disclosed herein. Moreover, insome embodiments, additional filtration or dialysis techniques known inthe art may be utilized prior to passing the blood from the targetsubject into the dialysis apparatus disclosed herein. In still someembodiments, methods of removing non-polar, hydrophobic and/or proteinbound uremic toxins from a target subject comprising using a dialysisapparatus may further comprise using a priming solution that is passedthrough the cartridge prior to use. The solution includes one or morelipocalins that would attach to the dialysis cartridge membrane asdescribed with respect to the self-contained cartridge including thelipocalins and other components as described above.

In some embodiments the methods disclosed herein may further comprisethe step of passing the blood through the dialysis apparatus such thatthe blood comes into contact with the at least one protein from thelipocalin family, which is bound to the blood side of the membrane. Insome embodiments, the membrane is a polysulfone membrane. Still in someembodiments, the method(s) may further comprise the step of passing theblood through the dialysis apparatus such that the blood comes intocontact with at least one OBP. In some embodiments, the OBP may be boundto a polysulfone membrane.

In some embodiments, the blood is passed through the polysulfonemembrane at an effective flow rate. The effective flow rate is,generally, the rate that allows for non-polar, hydrophobic, and/orprotein bound uremic toxins to bind to the at least one protein from thelipocalin family. The optimal flow rate to maximize the binding ofnon-polar toxins to the lipocalin proteins can be determined by one ofordinary skill in the art without undue experimentation. In embodimentswhere the polysulfone membrane comprises a plurality of proteins fromthe lipocalin family, the plurality of proteins removes the non-polar,hydrophobic, and/or protein bound toxins, thereby effectively reducingthe amount of or eliminating these toxins from the blood.

In some embodiments, the at least one lipocalin protein is selected fromOBP2A, OBP2B, and combinations thereof. In still other embodiments, theat least one polysulfone membrane may comprise a plurality of OBP2Abound thereon. In still other embodiments, the at least one polysulfonemembrane may comprise a plurality of OBP2B bound thereon. In still someembodiments, the at least one polysulfone membrane may comprise aplurality of OBP2A and OBP2B bound thereon.

In some embodiments, the dialysis apparatus or cartridge of the presentinvention is modified internally such that the flow of blood creates“edifies” and areas where the interface between the circulating bloodand the attached lipocalins is maximized. In still other embodiments,these dams and structural modifications are designed to maximize bindingfor specific combinations of lipocalins. In still other embodiments, thedialysis apparatus or cartridge of the present invention with specificcombinations of lipocalins, dams and blood flow specifications aredesigned to meet the metabolic needs of individual patients.Additionally, the methods disclosed herein may further include the stepsof removing the cleaned blood from the dialysis apparatus disclosedherein, passing the cleaned blood through a fluid flow path, and/orreturning the cleaned blood to the target subject. The cleaned blood maybe returned to the target subject via any suitable method known in theart, such as via a catheter. Additional filtration and/or dialysatetechniques as known in the art may be further administered prior topassing the cleaned blood back to the target subject.

Non-limiting examples of non-polar, hydrophobic, and/or protein bounduremic toxins that may be removed via the dialysis apparatus and methodsdisclosed herein include: p-cresol, p-cresyl sulfate, indoxyl sulfate,3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, hippuric acid,indoleacetic acid, and combinations thereof. Moreover, Vanholder et al.,“Review on uremic toxins: Classification, concentration, andinterindividual variability” Kidney International, Vol. 63, (2003), pp.1934-1943, which is incorporated by reference in its entirety herein,discloses uremic toxins which may be removed via the dialysis apparatusand/or methods disclosed herein. (See Vanholder et al., Table 1, Table2, and Table 3).

The non-polar uremic toxins removed by the dialysis apparatus or methodsdisclosed herein may exist as free substances or may bind to serumproteins, such as albumin. Accordingly, in some embodiments, thedialysis apparatus and methods disclosed herein are directed to removingat least one non-polar uremic toxin that is bound to a serum protein.For example, in some embodiments, this includes the removal of at leastone non-polar uremic toxin that is bound to albumin.

Further, disclosed herein is a method of removing at least one non-polaruremic toxin from the blood of a target subject comprising the steps ofpassing the blood of a target subject through a dialysis apparatus,wherein the dialysis apparatus comprises at least one polysulfonemembrane having at least one protein from the lipocalin family boundthereon, wherein the non-polar uremic toxin binds to the at least oneprotein form the lipocalin family and is removed from the blood of atarget patient. In some embodiments, the non-polar uremic toxin that isremoved is p-cresol. In still some embodiments, the at least onenon-polar uremic toxin is a free substance. In still some embodiments,the at least one non-polar uremic toxin is bound to at least one serumprotein.

The present disclosure further provides methods of preventing ortreating uremia including uremic clinical symptoms and side effects.Uremic clinical symptoms and side effects include, but are not limitedto, fatigue, anemia, itching, peripheral neuropathy, gastrointestinaldisorders including nausea, vomiting, diarrhea, cardiovascularcomplications including accelerated coronary and peripheral vasculardisease, left ventricular hypertrophy, cardiac fibrosis and acceleratedrates of arrhythmias. In some instances, the uremia arises fromhydrophobic, protein-bound uremic toxins.

Additionally, the present disclosure provides methods of utilizing oneor more lipocalin proteins described herein bound (e.g., specificbinding) in an apparatus of the present invention as described hereinfor the purpose of removing prescription and/or non-prescription drugsfrom a bodily fluid of a subject. In some embodiments, the presenceprescription or non-prescription drugs in the subject's bodily fluidresults from an accidental or intentional overdose. In some embodiments,the drug includes, but is not limited to, acetaminophen, phenytoin(Dilantin), lorazepam (Ativan), warfarin (Coumadin), tricyclicantidepressants, digoxin, theophylline, valproic acid, carbamazepine(Tegretol), and cocaine.

“Subject” as used herein is a subject in whom renal dialysis is neededor desired. A subject may be a patient. In some embodiments, the subjectis a human; however, a subject of this disclosure can include an animalsubject, particularly mammalian subjects such as canines, felines,bovines, caprines, equines, ovines, porcines, rodents (e.g. rats andmice), lagomorphs, primates (including non-human primates), etc.,including domesticated animals, companion animals and wild animals forveterinary medicine, treatment or pharmaceutical drug developmentpurposes.

The subjects relevant to this disclosure may be male or female and maybe any species and of any race or ethnicity, including, but not limitedto, Caucasian, African-American, African, Asian, Hispanic, Indian, etc.,and combined backgrounds. The subjects may be of any age, includingnewborn, neonate, infant, child, adolescent, adult, and geriatric.

In some embodiments, the subject has impaired kidney function, kidneydisease, acute kidney disease, chronic kidney disease, advanced kidneydisease or end stage renal disease (ESRD). In some embodiments, thesubject can have any type of primary disease that leads to impairedglomerular filtration rate (GFR) including, but not limited to diabetes,hypertension or glomerulonephritis.

In some embodiments, the subject is one in whom prescription ornon-prescription drug clearance or removal is needed or desired.Further, the subject can be a patient in a medical facility, hospitalsetting, ambulatory care setting, urgent or critical care setting,emergency medical services setting, drug rehabilitation setting, and thelike. In some embodiments, the subject may be suffering from acute orchronic prescription or non-prescription drug toxicity. In someembodiments, the subject has experienced an accidental or intentionalprescription or non-prescription drug overdose. In some embodiments, thesubject is one in whom medical management of prescription ornon-prescription drug overdose is desired.

All references cited in this specification, including withoutlimitation, all papers, publications, patents, patent applications,presentations, texts, reports, manuscripts, brochures, books, internetpostings, journal articles, periodicals, and the like, are herebyincorporated by reference into this specification in their entireties.The discussion of the references herein is intended merely to summarizethe assertions made by their authors and no admission is made that anyreference constitutes prior art. Applicants reserve the right tochallenge the accuracy and pertinence of the cited references.

Having now described the invention, the same will be illustrated withreference to certain examples, which are included herein forillustration purposes only, and which are not intended to be limiting ofthe invention.

EXAMPLES

Prophetic Experiments Confirming Efficacy of Lipocalin Modified DialysisCartridges

Experiment 1: Initial experiments will establish the concentrations ofP-cresol and where the fluorescence is linear and use this concentrationrange for the following prophetic experiments. Our initial experimentswill be determine the binding affinity, dissociation constants (Kd) andmolar stoichiometry of olfactory binding protein OBP-2a and OBP-2b andretinal binding protein-RBP-4 and other lipocalin molecules to freep-cresol, indoxyl sulfate, and other non-polar protein bound uremictoxins. The following table lists, but is not limited to uremic andprotein bound toxins that will be studied.

TABLE 1 Uremic and protein bound toxins Solute Group C_(N) C_(U) C_(Max)2-methoxyresorcinol phenols — 19.6 ± 81.2 322.0 3-deoxyglucosone AGE 0.3± 0.1 1.7 ± 1.0 3.5 CMPF 7.7 ± 3.3 61.0 ± 16.5 94.0 fructoselysine AGE —58.1 ± 10.8 79.7 glyoxal AGE 67.0 ± 20.0 221.0 ± 28.0  277.0 hippuricacid hippurates <5.0 247.0 ± 112.0 471.0 hydroquinone phenols <1.7 8.1 ±1.6 26.4 indole-3-acetic acid indoles — 50.6 ± 84.7 286.0 indoxylsulfate indoles 17.5 ± 17.5 875.0 ± 560.0 9076.9 kinurenine indoles 0.6± 5.4 53.0 ± 91.5 236.0 kynurenine indoles <391 686.4 ± 178.9 952.6kynurenic acid indoles <1.0 — 9.5 leptin peptides 8.4 ± 6.7 72.0 ± 60.6490.0 melatonin indoles 26.5 ± 7.1  175.8 ± 130.2 436.2 methylglyoxalAGE 47.0 ± 12.0 110.0 ± 18.0  146.0 N^(∈)-(carboxymethyl) AGE 1.1 ± 0.34.3 ± 1.3 6.9 lysine p-cresol phenols 0.6 ± 1.0 20.1 ± 10.3 40.7pentosidine AGE 51.6 ± 18.8 896.0 ± 448.0 2964.0 phenol phenols 0.6 ±0.2 2.7 ± 3.9 10.5 P-OHhippuric acid hippurates — 18.3 ± 6.6  31.5putrescine polyamines 21.1 ± 7.9  77.4 ± 27.3 132.0 quinolinic acidindoles  0.1 ± 0.05 1.5 ± 0.9 3.3 retinol-binding protein peptides <80192.0 ± 78.0  369.2 spermidine polyamines — 97.2 ± 45.0 187.2 sperminepolyamines — 18.2 ± 16.2 66.7CMPF=3-carboxy-4methyl-5-propyl-2-furanpropionic acid AGE=advancedglycation end products C_(N)=normal concentration (means ± SD or maximumvalue) C_(U)=mean/median uremic concentration (means ± SD or maximumvalue) C_(max)=maximal uremic concentration

Example 1

Using agarose beads bound with C-Myc antibodies, the capacity of OBPsand RBPs and all other lipocalins to displace p-cresol, p-cresol sulfateand all other polar and non-polar protein bound uremic toxins fromalbumin binding stores will be established. Studies to confirm thisobservation will be performed as follows.

In brief, agarose beads bound with anti-C-Myc antibodies will beincubated with C-myc labeled OBP2a, OBP2b and RBP-4. One ml of labeledagarose beads will to bind 7.0 nMoles of C-myc tagged lipocalins.

The C-Myc antibody tagged agarose beads will be loaded into SigmaSC-100-Sigma Prep Spin affinity columns and then washed with 400 ulphosphate buffered saline pH-7.4 containing 15mM Na+ azide aspreservative.

Sigma spin columns will be centrifuged at approximately 8200×g (10,000rpm in an Eppendorf 5415C microcentrifuge) for 1″ to remove wash bufferfollowed by the addition of 3.0 nMoles of C-Myc tagged OBP2a, OBP2b orRBP-4. Columns will be incubated at room 25° C. for 1 hour.

OBP-lipocalin labeled affinity columns will be incubated with increasingconcentration of p-cresol, p-cresol sulfate and indoxyl sulfate toconfirm the binding affinity of the lipocalins for the non-polar,protein bound uremic toxins. After thorough washing of the column of theeluents will be combined and the final concentration of p-cresoldetermined by Ultra-High Pressure Liquid Chromatography (U-HPLC). HPLCmethods are outlined in brief.

Ultra-High Pressure Liquid Chromatography will be performed as outlinedin Pretorius, C. J., McWhinney, B. C., Bilyana Sipinkoski, B., Johnson,L. A., Megan Rossi, M., Campbell, K., Ungerer, P. J. J. Reference rangesand biological variation of free and total serum 2 indoxyl- and p-cresolsulphate measured with a rapid UPLC 3 fluorescence detection method.Clin Chim Acta. 18; 419:122-6. 2013. Chemicals will be purchased fromSigma (USA):3-indoxyl-sulphate potassium salt (IS), p-cresol,4-ethylphenol, pyrimidine, diethyl ether and sodium-octanoate.Acetonitrile, ethanol, methanol and water were HPLC grade.Chromatography will be performed with a Waters Acquity UPLC I classsystem comprising of a binary solvent manager, flow through needleautosampler, fluorescence detector and column manager (Milford Mass.,USA) and an Acquity HSS T3 1.8 μm (2.1×50 mm) column with an Acquity BEHC18 1.7 μm VanGuard pre-column (2.1×5 mm). Mobile phase A will be 50mmol/L ammonium formate (pH 5.0) and mobile phase B was 100%acetonitrile. Mobile phase B increased with a linear gradient from 5 to25% over 2.1 min, and will be maintained isocratically at 70% for 0.4min, followed by 0.5 min at 99%. The column will be re-equilibrated withinitial conditions for 0.5 min. The load-ahead facility within thesystem will be enabled to minimize the run time. Injection volume willbe 2 μL for total IS (tIS) and total pCS (tpCS) and 5 μL for 95 fIS andfpCS samples. Column temperature was maintained at 45° C. IS eluted at1.5 min, pCS at 2.1 min and the internal standard at 2.65 min IS, pCSand the internal standard (50 μmol/L) 4-ethylphenol) will be quantifiedwith timed programmed fluorescence detection monitoring at specificexcitation/emission wavelengths (IS: 300/390 nm; pCS: 260/283 nm; and4-ethylphenol: 285/310 nm). Calibrators, quality control and samplepreparation calibrators, for tpCS and tIS will be prepared in salinespanning a range of 1 to 500 μmol/L and for fpCS and fIS of 0.1 to 100μmol/L. Quality control samples will be prepared by spiking pooled serumto obtain three levels as described in Table 1. Aliquots of the qualitycontrol materials will be stored at −80° C. fpCS and ffS measureddirectly, without addition of an internal standard, on ultrafiltratesprepared at room temperature from 200 μL of serum centrifuged for 10 minat 13,000 rpm with a 30,000 MWCO filter (Merck, Kilsyth, Australia). tISand tpCS will be measured after deproteinization of 100 μL, or albuminor serum solutions using 300 μL of ethanol containing internal standard(50 μmol/L 4-ethylphenol).The mixture will be centrifuged for 5 min at13,000 rpm and poured into a 2.0 mL tube that contained 200 μL H2O and 1mL dichloromethane. After vortexing for 1 min and centrifuging for 5 minat 13,000 rpm, 150 μL of the aqueous supernatant was transferred to aninjection vial. To compare ethanol vs. protein displacement, 100 μL ofserum was added to 100 μL of 0.25 M sodium octanoate (bindingcompetitor). After incubation at room temperature for 10 min, 600 μLethanol with internal standard will be added prior to extraction intodichloromethane.

Example 2

The above experiments will be extended to establish the ability ofOBP2a, OBP2b and RBP-4 and other lipocalins to remove p-cresol and otherprotein bound uremic toxins from albumin and other proteins in humanserum. Increasing concentrations of p-cresol will be incubated with aphysiologic concentration of human albumin (4.0 gm/dl) and thensubjected to affinity chromatography as outlined in steps 1-4. Thecolumn eluents, which will contain a mixture of free and bound p-cresol,will be separated using U-HPLC and the concentration of p-cresol thatdid not bind to the lipocalin column determined. These experiments willbe repeated using human serum and human albumin.

Example 3

Tertiary experiments will extend the observations of Steps 1-5 andexamine the role of pH on the binding affinity of OBPs and otherlipocalins for p-cresol and indoxyl sulfate. The rationale is that thehigh pH of bicarbonate dialysate could potentially alter the bindingaffinity of dialyzer bound lipocalins for p-cresol and other proteinbound uremic toxins. These experiments will determine whether lipocalinmodification of existing polysulfone dialyzers could lead to thecreation of a single dialysis cartridge capable of simultaneouslyclearing both water soluble and water-insoluble uremic toxins.

Methods: These experiments will be conducted in a manner similar toExperiment 1 and 2. In brief, the role of changing pH on the level ofp-cresol fluorescence that arises from binding to lipocalin proteinswill be examined. These experiments will be extended to include normaland human serum from patients with pre-dialysis CKD and stable ESRDpatients. In each of these latter experiments, the pH will be raisedfrom 6.0 to 8.0 and the level of fluorescence measured.

Example 5

These studies will calculate the optimal flow rates for removal ofp-cresol and other protein bound uremic toxins by OBPs and otherlipocalins. Binding capacities will be calculated from 50-500 mls/min.

Methods: In brief, histamine tagged lipocalins will be bound to nickelcoated columns. Varying concentrations of p-cresol in saline, albuminsolutions or human serum will run through the column at increasing flowrates; ranging from 5.0 ml/min up to a maximum of 500 mls/min. Thep-cresol bound OBPs will then be released from the column and the degreeof fluorescence measured as previously recorded.

Example 6

Using a porcine model of chronic kidney disease (Collaboration withMedical College of Georgia) to determine whether bulk removal ofnon-polar protein bound uremic toxins will block the degradation ofConnexin 43; a protein implicated in gap junction propagation ofexcitation potential across cardiac myocytes. The studies in Example 6will create a porcine model of chronic renal failure using unilateralnephrectomy and ⅔ contralateral nephrectomy. CKD pigs will be sacrificedat 3 months and the level of plasma p-cresol quantified by U-HPLC asdiscussed in paragraph [0068] correlated with the level of Connexin-43expression in cardiac myocytes. The rationale is that cell culturemodels have shown that at concentrations commonly found among ESRDpatients, p-cresol can induce a dose-dependent degradation ofConnexin-43 within cardiac gap junctions. Connexins, or gap junctionproteins, are a family of structurally related transmembrane proteinsthat assemble to form gap junctions. Each gap junction is composed oftwo hemichannels, or connexons, which is composed of six connexinmolecules. Connexins in one cell connect with connexins in adjacentcells across the gap junction, connecting the cells electrically. Atrialfibrillation (AF) can be associated with a change in the number ofconnexins between cells. AF can also be associated with a change inconnexin distribution, particularly lateralization of connexins SeePeng, Y. S., Ding, H. S., Lin, Y. T., Syu, J. P., Chen, Y., Wang, S. M.Uremic toxin p-cresol induces disassembly of gap junctions ofcardiomyocytes Toxicology 302 (2012) 11-17.

These experiments will be extended by studying four animal groups:Group-1 normal controls, Group-2 CKD pigs without intervention, Group-3CKD pigs treated with daily plasma phoresis for 1 week and Group-4 CKDpigs treated with daily plasmaphoresis and receiving daily infusion ofp-cresol. Because p-cresol rapidly degrades Connexin-43 and the loss ofthis protein contributes to atrial arrhythmias, it is believed thatGroup-3 subjects will exhibit Connexin-43 levels that approximate thoseof the normal controls.

Example 7

These studies will build an experimental parallel plate dialyzerprototype of the CLEAR cartridge where the process of covalently bindingproteins from the lipocalin family to sheets of polysulfone materialwill be refined. The plate dialyzer technology will be applied to theCKD pig models where the biologic endpoint of p-cresol removal will berestoration of Connexin-43 expression in cardiac gap junctions. Theseexperiments will be extended to creation of a CLEAR cartridge usingpolysulfone hollow fiber dialysis membranes.

Example 8

These studies will extend the findings of Example 7 and a Phase I trialto determine the safety and efficacy of the CLEAR cartridge in patientswith ESRD will be conducted.

Example 9

These studies will include a randomized prospective trial comparingconventional hemodialysis with hemodialysis and the CLEAR cartridgetechnology in the following clinical complications of End Stage RenalDisease (ESRD).

-   -   1) Rate of post dialysis bradycardia and incidence of Atrial or        Ventricular arrhythmia.    -   2) Determining the rate of development of Left ventricular        hypertrophy (LVH) and diastolic dysfunction in incident dialysis        patients    -   3) Determine the efficacy of the CLEAR cartridge technology on        the incidence and severity of uremic Pruritis    -   4) Determine the efficacy of the CLEAR cartridge technology on        cognition, rates of depression and other neuropsychiatric        complications of ESRD    -   5) Determine the efficacy of the CLEAR cartridge technology on        olfactory capabilities in ESRD patients    -   6) Determine the efficacy of the CLEAR cartridge technology in        reducing the rate of malaria infection in ESRD that are in        countries with endemic populations of malaria.

Prophetic Experiments Confirming Efficacy of Lipocalin Modified DialysisCartridges Binding and Clearance of Protein Bound Poisons and DrugOverdoses

Example 1

To expand the utility of a lipocalin modified dialysis cartridge, wewill perform preliminary experiments documenting the ability oflipocalins as described herein, and in particular, OBP2a, OBP2b, RBP4,and LFABP, to bind and remove highly protein bound drugs from theplasma. The principal clinical application of this extension is the useof the CLEAR cartridge to remove potentially life threatening drugoverdoses. Drugs that will be tested for lipocalin binding and drugremoval include, but are not limited to the following drugs,acetaminophen, phenytoin (Dilantin), lorazepam (Ativan), warfarin(Coumadin), tricyclic antidepressants, digoxin, theophylline, valproicacid, carbamazepine (Tegretol), and cocaine.

The molar to molar binding affinity of the above drugs that have beenconsistently reported to be a cause of drug overdose will beinvestigated for their ability to bind and be cleared by lipocalinsbound to a membrane as described herein. The same methods used in theexperiments described above will be used to confirm the binding of drugsto the CLEAR cartridge at flow rates compatible for conventionaldialysis machines.

Although embodiments of the disclosure have been described usingspecific terms, devices, and methods, such description is forillustrative purposes only. The words used are words of descriptionrather than of limitation. It is to be understood that changes andvariations may be made by those of ordinary skill in the art withoutdeparting from the spirit or the scope of the present disclosure, whichis set forth in the following claims. In addition, it should beunderstood that aspects of the various embodiments may be interchangedin whole or in part. Therefore, the spirit and scope of the appendedclaims should not be limited to the description of the versionscontained therein.

What is claimed is:
 1. An apparatus for a dialysis procedure comprisinga body having an inlet and an outlet and defining an interior, theinterior including at least one membrane having at least one proteinfrom the lipocalin family bound thereon, wherein the apparatus isarranged so that a fluid entering the apparatus contacts the at leastone protein from the lipocalin family bound to the at least one membranein the interior of the apparatus, wherein the at least one protein fromthe lipocalin family comprises at least one olfactory binding protein.2. The apparatus of claim 1, wherein the membrane is at least one ofpolyamide, polysulfone, polyethersulfone, cellulose acetate, triacetate,polyacrylonitrile or polymethylmethacylate.
 3. The apparatus of claim 1,wherein the at least one membrane is a polysulfone membrane.
 4. Theapparatus of claim 1, wherein the at least one protein is covalentlybound to the at least one membrane.
 5. The apparatus of claim 1, whereinthe at least one olfactory binding protein is selected from the groupconsisting of OBP2A, OBP2B and combinations thereof.
 6. The apparatus ofclaim 1, wherein the clearance of hydrophobic, protein bound uremictoxins is facilitated.
 7. The apparatus of claim 6, wherein thehydrophobic, protein bound uremic toxin is at least one of p-cresol,indoxyl sulfate or p-cresol sulfate.
 8. A method of dialysis comprisingsubjecting a bodily fluid from a subject to an apparatus comprising abody having an inlet and an outlet and defining an interior, theinterior including at least one membrane having at least one proteinfrom the lipocalin family bound thereon, wherein the apparatus isarranged so that the bodily fluid entering the apparatus contacts the atleast one protein from the lipocalin family bound to the at least onemembrane in the interior of the apparatus, wherein the at least oneprotein from the lipocalin family comprises at least one olfactorybinding protein.
 9. The method of claim 8, wherein the at least onemembrane is at least one of polyamide, polysulfone, polyethersulfone,cellulose acetate, triacetate, polyacrylonitrile orpolymethylmethacylate.
 10. The method of claim 8, wherein the at leastone membrane is a polysulfone membrane.
 11. The method of claim 8,wherein the at least one protein is covalently bound to the at least onemembrane.
 12. The method of claim 8, wherein the at least one olfactorybinding protein is selected from the group consisting of OBP2A, OBP2Band combinations thereof.
 13. The method of claim 8, wherein theclearance of hydrophobic, protein bound uremic toxins is facilitated.14. The method of claim 13, wherein the hydrophobic, protein bounduremic toxin is at least one of p-cresol, indoxyl sulfate or p-cresolsulfate.
 15. A method of preventing or treating uremia comprisingsubjecting a bodily fluid from a subject to an apparatus comprising abody having an inlet and an outlet and defining an interior, theinterior including at least one membrane having at least one proteinfrom the lipocalin family bound thereon, wherein the apparatus isarranged so that the bodily fluid entering the apparatus contacts the atleast one protein from the lipocalin family bound to the at least onemembrane in the interior of the apparatus, wherein the at least oneprotein from the lipocalin family comprises at least one olfactorybinding protein.
 16. The method of claim 15, wherein the at least onemembrane is at least one of polyamide, polysulfone, polyethersulfone,cellulose acetate, triacetate, polyacrylonitrile orpolymethylmethacylate.
 17. The method of claim 15, wherein the at leastone membrane is a polysulfone membrane.
 18. The method of claim 15,wherein the at least one protein is covalently bound to the at least onemembrane.
 19. The method of claim 15, wherein the at least one olfactorybinding protein is selected from the group consisting of OBP2A, OBP2Band combinations thereof.
 20. The method of claim 15, wherein theclearance of hydrophobic, protein bound uremic toxins is facilitated.21. The method of claim 20, wherein the hydrophobic, protein bounduremic toxin is at least one of p-cresol, indoxyl sulfate or p-cresolsulfate.
 22. The method of claim 15, wherein the uremia is characterizedby at least one of fatigue, anemia, itching, peripheral neuropathy, agastrointestinal disorder, nausea, vomiting, diarrhea, cardiovascularcomplications, coronary vascular disease, peripheral vascular disease,left ventricular hypertrophy, cardiac fibrosis and accelerated rates ofarrhythmias.
 23. The method of claim 15, wherein the uremia is caused byhydrophobic, protein-bound uremic toxins.
 24. A method of dialysiscomprising: (a) providing a solution comprising at least one proteinfrom the lipocalin family to the interior of a dialysis apparatus; and(b) subjecting a bodily fluid from a subject to the dialysis apparatuscomprising a body having an inlet and an outlet and defining aninterior, wherein the interior includes at least one membrane capable ofbinding the at least one protein from the lipocalin family and thedialysis apparatus is arranged so that the bodily fluid entering thedialysis apparatus contacts the at least one protein from the lipocalinfamily bound to the least one membrane in the interior of the apparatus,wherein the least one protein from the lipocalin family comprises atleast one olfactory binding protein, and wherein clearance ofhydrophobic , protein bound uremic toxins is facilitated.
 25. A methodof extracorporeal detoxification comprising subjecting a bodily fluidfrom a subject to an apparatus comprising a body having an inlet and anoutlet and defining an interior, wherein the interior includes at leastone membrane capable of binding the at least one protein from thelipocalin family and the apparatus is arranged so that the bodily fluidentering the apparatus contacts the at least one protein from thelipocalin family bound to the at least one membrane in the interior ofthe apparatus, wherein the at least one protein from the lipocalinfamily comprises at least one olfactory binding protein, whereinclearance of a drug is facilitated.
 26. The method of claim 25, whereinthe drug is a prescription or a non-prescription drug.
 27. The method ofclaim 25, wherein the detoxification concerns an accidental orintentional drug overdose.